GSM6021756: AAVil6_high_retina_1; Mus musculus; RNA-Seq
Flash frozen dissected retina and eye cup tissues (n=72) were homogenized in 700 µL Qiazol (Qiagen) using a Precellys Evolution homogenizer (Bertin Technologies). Retina samples were homogenized by ceramic beads (MPbio, 6913-500) and eye cup samples by metal beads (Bertin Technologies, 15987602). Total RNA was extracted using the miRNeasy Micro Kit (Qiagen, 217084) according to the manufacturer's tissue protocol including on-column DNase treatment (Qiagen, 79254). Total RNA samples (36 eye cup, 36 retina) were quantitatively and qualitatively assessed using the fluorescence-based Broad Range Quant-iT RNA Assay Kit (Thermo Fisher Scientific) and the Standard Sensitivity RNA Analysis DNF-471 Kit on a 96-channel Fragment Analyzer (Agilent), respectively. Retina- and eye cup-derived RNA samples were normalized on the MicroLab STAR automated liquid platform (Hamilton). Total RNA input of 250ng was used for library construction with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina #E7760, together with the NEBNext Poly(A) mRNA Magnetic Isolation Module #E7490 upstream and the NEBNext Multiplex Oligos for Illumina #E7600 downstream (all New England Biolabs). The only deviation from the manufacturer's protocol was the use of Ampure XP beads (Beckman Coulter) for double-stranded cDNA purification, instead of the recommended SPRIselect Beads. The index PCR was performed with 12 cycles, while the final library was eluted in 35µL. mRNA libraries were then quantified by the High Sensitivity dsDNA Quanti-iT Assay Kit (ThermoFisher) on a Synergy HTX (BioTek). Library molarity averaged at 149nM. mRNA libraries were also assessed for size distribution (smear analysis of 360bp average) and adapter dimer presence (<0.5%) by the High Sensitivity Small Fragment DNF-477 Kit on a 96-channel Fragment Analyzer (Agilent). All 70 sequencing libraries were then normalized on the MicroLab STAR (Hamilton), pooled and spiked in with PhiX Control v3 (Illumina). The library pool was subsequently clustered on an S4 Flow Cell and sequenced on a NovaSeq 6000 Sequencing System (Illumina) with dual index, paired-end reads at 2 x 100 bp length.